Whooping cough in humans is caused by members of the Bordetella species, especially B. pertussis. Following disease or vaccination, antibodies are elicited against several bacterial proteins, especially pertussis toxin (PT), filamentous haemagglutinin (FHA), the 69kDa outer membrane protein (69kD or pertactin) and the fimbrial agglutinogens. PT is a major protective antigen and is also associated with virulence. These antigens have been proposed to formulate single or multi-component pertussis vaccines and, as such, their efficient production and purification are essential.
During fermentation of B. pertussis, it has been found that FHA is secreted at 7 and 10 times the molar levels of 69kD or PT, respectively. Therefore, PT and pertactin are limiting antigens in the production of a component vaccine. Furthermore, the relative overproduction of FHA makes the purification of other antigens expressed at lower levels more difficult. This is especially true for PT, which may be initially co-purified with FHA in some purification protocols. It is also of concern that active PT may contaminate the FHA preparation, which then needs to be chemically detoxified to inactivate the residual native toxin. In the case of genetically detoxified pertussis toxin (described in published EPO patent application No. 0322115 and corresponding U.S. Pat. No. 5,085,862, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference; Loosmore et al., Infect. Immun. 58, 3653 [1990]), this would not be a problem. In either case, it would be advantageous, from a vaccine production viewpoint, to have a B. pertussis strain which does not produce FHA and/or one which does not produce PT. For example, an FHA.sup.- strain in which the FHA gene has been deleted could be used to produce all other antigens under optimized fermentation and purification conditions. Similarly, a PT.sup.- strain would produce FHA with no possibility of contamination by pertussis toxin.
There are B. pertussis gene products which are not necessary in a whooping cough vaccine, for example, dermonecrotic toxin (DNT) and possibly adenylate cyclase toxin. The gene loci coding for such products could be considered to be dispensable in a vaccine strain as long as the strain remains viable and produces the other antigens in satisfactory amounts. The deletion of dispensable genes offers the possibility of further genetic manipulation by in situ gene insertion at the loci of gene deletions. For example, the adenylate cyclase gene (CYA) could be deleted and one or more additional copies of one of the candidate vaccine antigen genes inserted at the CYA locus. The additional gene(s) may be introduced through homologous recombination using the flanking sequences of the deleted gene to direct in situ gene insertion. Alternatively, gene copies also may be introduced through random insertion into the chromosome or through the introduction of replicating plasmids. Multiple copies of regulatory genes, such as the vir regulon, Bvg, could also be introduced to modify antigen expression.
Examples of deleted strains could be TOX.sup.-, FHA.sup.-, PRN.sup.-, CYA.sup.-, DNT.sup.- or any combination of these or other gene deletions. Inserted genes could be any of the TOX, FHA, PRN or any other native, mutated, or hybrid genes and one or more copies could be inserted in tandem or separately. Such genetic manipulations aimed at augmenting a gene copy number with or without deletion of other genes, could lead to substantial enhancement of Bordetella antigen production and/or optimization of antigen purification.
Lee et al (1989, Infect. Immun. 57: 1413-1418) attempted to generate hypertoxigenic strains of B. pertussis by expression of the TOX operon from a multicopy replicating plasmid. No increase in PT production was observed, however, and the plasmid was rearranged, the TOX operon deleted or the transconjugants underwent conversion to an avirulent phase.